An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility

  1. Fernanda M Bosada
  2. Karel van Duijvenboden
  3. Alexandra E Giovou
  4. Mathilde R Rivaud
  5. Jae-Sun Uhm
  6. Arie O Verkerk
  7. Bastiaan J Boukens
  8. Vincent M Christoffels

  • Curated by eLife

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    Evaluation Summary:

    This manuscript presents an interesting and informative study on two regulatory elements found near atrial fibrillation-associated regions and their effect on Tbx5 expression and arrhythmia susceptibility in a mouse model. The multilevel approaches and analyses are rigorous, and the conclusions are justified by the data. Tbx5 expression may be of relevance for human atrial fibrillation and disease risk in patients, and the work is of potential interest to scientists in the fields of gene dosage, gene regulation, genetic susceptibility, genetic variants and cardiovascular biology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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Abstract

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)) and one downstream (RE(down)) of Tbx5 . In both lines, we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial Tbx5 expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in Tbx5 RE(int)KO cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in Tbx5 RE(int)KO mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

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  1. Version published to 10.7554/elife.80317 on eLife
  2. eLife

    Evaluation Summary:

    This manuscript presents an interesting and informative study on two regulatory elements found near atrial fibrillation-associated regions and their effect on Tbx5 expression and arrhythmia susceptibility in a mouse model. The multilevel approaches and analyses are rigorous, and the conclusions are justified by the data. Tbx5 expression may be of relevance for human atrial fibrillation and disease risk in patients, and the work is of potential interest to scientists in the fields of gene dosage, gene regulation, genetic susceptibility, genetic variants and cardiovascular biology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Bosada et al present a study on how regulatory elements found in two atrial fibrillation-associated regions at the TBX5 locus correlate to Tbx5 expression levels and arrhythmia susceptibility. In transgenic mouse models, the investigators deleted the orthologues of these regulatory elements at the human Tbx5 locus. Tbx5 expression levels were increased in both models, and the downstream impact on epigenetic and gene expression levels was assessed. The RE(int)-/- mice had higher expression levels of Tbx5 compared to RE(down)-/- mice and this was correlated with increased atrial arrhythmia inducibility and higher numbers of transcripts impacted in the atrial gene regulatory network analysis. Multiple pathways are affected, and the authors present data on the interaction between Tbx5 and Prrx1, which encodes a cardiac transcription factor and the human ortholog harbors an atrial fibrillation-associated variant. The presented work links with the prior observation that increase in Tbx5 expression is associated with human atrial fibrillation and provides a plausible mechanistic link.

  4. eLife

    Reviewer #2 (Public Review):

    Bosada et al. investigated how non-coding risk variants associated with atrial fibrillation (AF) might affect putative enhancers that potentially regulate gene expression and dosage of a disease-associated transcription factor (TF) Tbx5, to cause atrial arrhythmias in mouse hearts.

    The authors integrate multiple types of genetic and genomic analysis, including GWAS, in humans for identifying putative response elements potentially vulnerable to disease-associated variants, that regulate TBX5 expression. AF-associated risk variants in RE(int) were tested and one displayed increased enhancer activity by luciferase assay in transient transfections of HL-1 cells. The authors then identify syntenic regions in mouse. Targeted deletion of each region demonstrates an increase in Tbx5 gene expression, but not other nearby genes within the same TAD, in the heart. Some degree of arrythmia predisposition in vivo was observed. This was shown at the level of atrial function, as well as for atrial cardiomyocytes. Further, discovery of a TBX5-dependent pathway from increase in Tbx5 uncovers a genetic interaction between Tbx5 and Prrx, inferred from targeted mutations of enhancers that regulate each TF, linking risk variants of two TFs associated with AF.

    This work provides further insights for the observations that an increase in TBX5 dosage or a gain-of-function mutation of TBX5 have been associated with AF in humans. These findings uncover potential mechanisms to maintain proper gene dosage, which is essential for cardiac rhythm control.

    The work presented is rigorous, and the results clearly support the conclusions.

  5. eLife

    Reviewer #3 (Public Review):

    In the manuscript by Bosada et al, the authors present work identifying and interrogating two cis-regulatory elements at TBX5 associated with atrial fibrillation. Mouse models lacking both copies of either element, but particularly one in the last intron of the Tbx5 gene, referred to as RE(int), results in increased Tbx5 expression, changes to cardiac electrophysiology, and downstream gene expression changes. Of interest, RE(int) induces expression of Prrx1, also associated with atrial fibrillation, and compound mutants partially rescues the RE(int) phenotype. Overall, this paper is of interest and advances our understanding of TBX5 in atrial fibrillation risk in humans. Critically, this study focuses on the impact of risk SNPs, which increase TBX5 expression in patients, while previous studies involving TBX5 in atrial fibrillation have focused on decreased expression.

    The authors' work presents the following major claims:
    Figure 1. Identification of two, independently segregating risk regions at TBX5, which are conserved in humans and mice with predicted cis-regulatory functions termed RE(int) and RE(down).
    Figure 2. Homozygous RE(int) and RE(down) mutants, with a particular focus on RE(int) mutants, resulting in increased expression of Tbx5. The functional SNP appears to be rs7312625 A>G.
    Figure 3. Homozygous RE(int) mutants demonstrate several cardiac electrophysiological changes consistent with increased atrial fibrillation risk in humans.
    Figure 4. Cellular electrophysiology of RE(int) mutant cardiomyocytes demonstrates additional supportive changes to explain whole organ phenotypes presented in Figure 3.
    Figure 5. Transcriptional profiling of RE(int) and RE(down) homozygous mutants demonstrates many significant differences from control samples that are suggestive of specific mechanisms disrupting cardiac electrophysiology, including calcium and potassium regulators and gap junctions.
    Figure 6. Homozygous RE(int) and Prrx1(enh) mutant alleles genetically interact and result in partial rescue of phenotypes from RE(int) alone.

  6. Version published to 10.1101/2022.05.14.491627v1 on bioRxiv